Western Blotting

After proteins are run on an SDS-PAGE gel, a transfer is executed. What is the purpose of the transfer in Western blot protocol?

• Denature the proteins in the sample

• Visualize the proteins run on the gel

• Move proteins from the SDS-PAGE gel to a nitrocellulose membrane

• Probe the gel with an antibody to detect a protein of interest

Why is it called immunoblott?

• because it involves human antibody

• because the membrane contains antibody

• because it applies fluorescent antibodies

• because it applies antibody to tag protein

• because the protein of interest is an antibody

Which is the right orientation?

• cathode - filter paper - gel - transfer membrane - filter paper - anode

• cathode - filter paper - transfer membrane - gel - filter paper - anode

What is the purpose of washing in every step in western blotting?

• to ensure a stable pH environment in the tank

• to avoid protein degradation

• to remove any unbound antibodies

• to moisten the transfer membrane

• to remove any contaminating residues

State the purpose of blocking

• to block nonspecific antibody from binding to protein

• to avoid non specific binding of antibody

• to avoid antibodies from binding to protein

• to prevent protein from being washed off

• to prevent unwanted contamination towards proteins

Primary antibody targets..

• nitrocellulose surface

• specific protein

• primary antibody

• conjugated secondary antibody

• fluorochrome

Conjugated secondary antibody targets..

• nitrocellulose surface

• specific protein

• primary antibody

• conjugated secondary antibody

• fluorochrome